Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale
N C P Cross,1,2 H E White,1,2 T Ernst,3 L Welden,4 C Dietz,5 G Saglio,6 F-X Mahon,7 C C
Wong,8,* D Zheng,8 S Wong,8 S-S Wang,8 S Akiki,9 F Albano,10 H Andrikovics,11,12 J Anwar,13 G Balatzenko,14 I Bendit,15 J Beveridge,16 N Boeckx,17,18 N Cerveira,19 S-M Cheng,20 D
Colomer,21 S Czurda,22 F Daraio,23 S Dulucq,24 L Eggen,25 H El Housni,26 G Gerrard,27 M Gniot,28 B Izzo,29,30 D Jacquin,31 J J W M Janssen,32 S Jeromin,33 T Jurcek,34 D-W Kim,35 K Machova-Polakova,36 J Martinez-Lopez,37 M McBean,38 S Mesanovic,39 G
Mitterbauer-Hohendanner,40 H Mobtaker,31 M-J Mozziconacci,41 T Pajič,42 N Pallisgaard,43 P Panagiotidis,44 R D Press,45 Y-Z Qin,46 J Radich,47 T Sacha,48 T Touloumenidou,49 P Waits,50 E Wilkinson,51 R Zadro,52 M C Müller,5 A Hochhaus,3 and S Branford4,53,54,55
Abstract article-meta
Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment
cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR1–MR4), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR4.5 level. The secondary panel was
calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR4.5 sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.
Go to: Introduction
The development of BCR-ABL1 tyrosine kinase inhibitors, from the first-generation imatinib to newer agents such as nilotinib and dasatinib, has enabled progressively deeper molecular responses in chronic myeloid leukemia (CML) patients undergoing tyrosine kinase inhibitor therapy.1, 2 Deeper molecular responses are defined as BCR-ABL1 levels of ⩽0.01% (MR4) and ⩽0.0032% (MR4.5) on the
international reporting scale (International Scale (IS)) and are important milestones for patients
considering treatment cessation.3 Other landmarks on the IS also represent different treatment decision thresholds and prognostic outcomes.4 For example, patients who reach 10% IS or below at 3 months after treatment have significantly higher rates of MR4.5 by 5 years,5 and reaching 0.1% IS (major molecular response) by 12 months of treatment is predictive of subsequently achieving undetectable BCR-ABL1 levels.6 Thus, regular molecular monitoring using real-time reverse-transcription
quantitative PCR (RT-qPCR) is recommended for optimal disease management, and treatment decisions rely on achieving milestone molecular responses in the first year of therapy and beyond.2, 7 As treatment decisions are directly impacted by test results, accuracy and precision of BCR-ABL1 assays across the entire measurement range is crucial for patient management, especially in patients with deep molecular responses when considering possible treatment cessation. It is well known that high variability exists between RT-qPCR methods used in different laboratories.8, 9 The first
international standardization attempt occurred in 2003, when different BCR-ABL1 assays used in the International Randomised Study of Interferon versus STI571 (IRIS) trial established IS based on 30 CML patient samples.10 Subsequently, a process for establishing a test-specific IS conversion factor (CF) by exchanging 20–30 CML patient samples with a reference laboratory was developed.11 Although this process works well for laboratories with tests that show good stability over time, it is time consuming, expensive and difficult to access for smaller laboratories.12, 13
In 2010, the ‘first International Genetic Reference Panel for quantitation of BCR-ABL mRNA' was developed as a primary standard for BCR-ABL1 assay IS calibration and accredited by the World Health Organization (WHO).14 The WHO panel is made of lyophilized K562 and HL-60 cell line mixtures, which allows the inclusion of cellular RNA extraction in the IS calibration against the two major BCR-ABL1 breakpoints (e13a2 and e14a2) and carries three sets of nominal %BCR-ABL1 values using ABL1, BCR and GUSB as reference genes. Owing to restricted access, the WHO panel is currently only available to manufacturers of BCR-ABL1 test kits and secondary standards.13 The commercial secondary standards available to date are made of RNA;15, 16 thus, RNA extraction is not included in the IS calibration process, except when the standards are artificially spiked into the cell samples. Furthermore, none of these are calibrated to the WHO panel against all three reference genes. In this study, we describe the successful development of the first cell-based BCR-ABL1 secondary reference panel that is traceable to and faithfully replicates the WHO panel in both raw materials (lyophilized K562 and HL-60 cell mixes) and manufacturing process, with the addition of a MR4.5 level. Nominal %BCR-ABL1 IS values were assigned to the secondary panel using
reverse-transcription droplet digital PCR (RT-ddPCR) against ABL1, BCR and GUSB. The secondary panel was successfully evaluated by 45 different BCR-ABL1 assays in a subsequent international multi-center evaluation study.
Go to: Materials and methods
Manufacturing and IS calibration of secondary reference panel
K562 (ATCC CCL-243) and HL-60 cells (ATCC CCL-240) (American Type Culture Collection, Manassas, VA, USA) were cultured, mixed and lyophilized following methods described by White et al.14 with minor modifications (Supplementary Information). Calibration to the WHO standards was performed as described.14 IS calibration using ABL1 as a reference gene was conducted using 10 sets of WHO ‘first International Genetic Reference Panel for quantitation of BCR-ABL mRNA' panels (National Institute for Biological Standards and Control, South Mimms, UK). Calibration using BCR and GUSB was conducted in a second study using another 10 sets of WHO panels. On each day of 10 non-consecutive days, 1 WHO panel and 2–3 secondary panels were tested using RT-ddPCR in 4 replicates for the MR1 (10% BCR-ABLIS) to MR4 (0.01% BCR-ABLIS) samples and in 8 replicates for the MR4.5 (0.0032% BCR-ABLIS) sample, to enhance assay precision. Data analysis was
performed using the statistical methods described by White et al.14 Reverse-transcription droplet digital PCR
RNA extraction from the secondary panel was performed using RNeasy mini kits (Qiagen, Hilden, Germany). Reverse transcription was performed using ABI High Capacity cDNA reverse-transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) and ddPCR was performed using 2X ddPCR Supermix (Bio-Rad, Hercules, CA, USA) on the QX-100 or QX-200 ddPCR system (Bio-Rad). All primer and probe sequences are listed in Table 1. BCR-ABL1 and reference genes were run as
singleplex reactions in separate wells. To achieve optimal assay precision and avoid signal saturation, cDNA input for BCR-ABL1 per 20 μl reaction was 80 ng for the MR1 sample, 400 ng for the MR2 sample and 675 ng for samples ⩽MR3. cDNA input for reference genes was 10 ng per 20 μl reaction for all three reference genes. For each RT-ddPCR run, wells with >9025 accepted droplets were considered valid as per the manufacturer's recommendations.
Results
Manufacturing and IS calibration of the WHO BCR-ABL1 secondary reference panel
We successfully manufactured >12 000 vials of secondary BCR-ABL1 lyophilized cell reference panel, using the same K562 and HL-60 cell lines and following similar manufacturing procedures as the primary WHO panel (Supplementary Information).14 An MR4.5 level was added to the secondary panel, to enable more accurate IS calibration at this critical level, as CML patients reaching this deep molecular response are increasingly being considered for treatment cessation. Quality-control
assessments indicated that the secondary panel had minimal residual moisture, excellent vial-to-vial homogeneity and >2.5 years of real-time stability (Supplementary Information).
To calibrate the secondary panel to the WHO ‘first International Genetic Reference Panel for
quantitation of BCR-ABL1 mRNA', we followed the study design described by White et al.,14 except that the sample size was doubled to strengthen the statistical power (Supplementary Information). RT-ddPCR was chosen as the calibration method, owing to its superior sensitivity, precision and absolute quantification capability compared with RT-qPCR.17, 18 At the time of this study, no commercially available BCR-ABL1 test used BCR or GUSB as reference genes. We developed three sets of RT-ddPCR assays, including BCR-ABL1/ABL1, BCR-ABL1/BCR and BCR-ABL1/GUSB, to enable IS calibration of the secondary panel against all three reference genes. All RT-ddPCR assays were validated following a combination of industry best practices, Minimum Information for Publication of Quantitative Real-Time PCR Experiments guideline, and Clinical and Laboratory Standards Institute guideline, to ensure proper accuracy, precision, sensitivity and linearity were achieved (Supplementary Information).19, 20, 21, 22 Using methods described by White et al.,14 we determined the IS CF for the RT-ddPCR assays to be 0.93 for ABL1/ABL1, 1.85 for ABL1/BCR and 1.28 for BCR-ABL1/GUSB.
Each CF was subsequently applied to the empirical %BCR-ABL1 of the secondary panel measured by RT-ddPCR, to obtain the assigned %BCR-ABL1IS values (Figure 1 and Table 2a). We found that the mean %BCR-ABL1 for each level of the secondary panel met all targeted BCR-ABL1 levels and were within 1.3-fold of the WHO standards values. The assigned %BCR-ABL1IS of level E was 0.0038%, 0.0050% and 0.0029% for ABL1, BCR and GUSB, respectively, indicating that an MR4.5 level was successfully created. Moreover, the mean copy number of BCR-ABL1, ABL1, BCR and GUSB per ng of RNA measured using RT-ddPCR was highly similar between the WHO and secondary panels (Table 2b). This demonstrated that the secondary panel replicated the primary WHO panel faithfully, with the successful addition of an MR4.5 level.
